This NIH mentored Career Development Award proposal describes a five year training program for the candidate, a physician scientist with the long-term goal of becoming an independent academic investigator with a research focus on epithelial ion transport related to kidney physiology and diseases. To accomplish these goals the candidate and his mentors developed an integrated plan encompassing novel scientific ideas, advanced training in basic science research and a detailed career development plan. The candidate proposes to build on a background in basic research developed during his postdoctoral research and fellowship training, specifically on his previous findings in genetics and expression of Uromodulin (UMOD), a protein which if altered causes kidney stones and hypercalciuria in mice. Most of the tubular Ca2+ is reabsorbed in the proximal tubule by paracellular mechanisms. However, in the distal convoluted tubule (DCT) and connecting tubule (CNT) Ca2+ is absorbed via transepithelial transport by the Ca2+ channel TRPV5. Urinary analysis in mutant Umod mice suggests impaired calcium absorption in the DCT and CNT where TRPV5 is the most abundant calcium channel. TRPV5 is regulated by Klotho, an anti-aging protein. Klotho functions as a sialidase, exposing galactose-N-acetylglucosamine at N-glycosylated TRPV5 channels, which then bind to galectin-1, a ubiquitous lectin. This results in impairment of TRPV5 endocytosis and increased TRPV5 surface abundance. My preliminary data indicate that UMOD upregulates TRPV5 and that UMOD and Klotho work in the same pathway in this respect. This proposal seeks to improve the understanding how UMOD interferes with renal Ca2+ absorption. My aims are to analyze what the molecular mechanism of TRPV5 upregulation by UMOD is; to examine the physiological role of UMOD in regulating renal Ca2+ excretion in vivo; and to examine the mechanism of TRPV5 co-regulation by UMOD and Klotho and test the significance in vivo. I will test my hypotheses utilizing electrophysiology, protein biochemistry and mouse genetics. I will analyze how UMOD increases TRPV5 activity by testing UMOD's impact on TRPV5 endocytosis, TRPV5 single channel open probability and conductance. I will test if decreased secretion of mutant UMOD may contribute to less TRPV5 activation thus causing hypercalciuria. I will test the significance of my in vitro findings in viv by analyzing TRPV5 expression and activity in isolated native tubules from mutant Umod and wild type animals. I will examine the mechanism how UMOD and Klotho co-regulate TRPV5. To analyze if UMOD and Klotho regulate renal Ca2+ absorption together in vivo I will test if Klotho overexpression can rescue hypercalciuria due to mutant UMOD. Crossed mutant Umod; overexpressing Klotho mice will be analyzed for renal Ca2+ excretion, TRPV5 expression and activity in the DCT/CNT. Results obtained by these experiments will improve the knowledge of hypercalciuria, a significant risk factor for kidney stones, and may contribute to the development of novel treatment strategies in nephrolithiasis.